NF-kappaB- and C/EBPbeta-driven interleukin-1beta gene expression and PAK1-mediated caspase-1 activation play essential roles in interleukin-1beta release from Helicobacter pylori lipopolysaccharide-stimulated macrophages

Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1beta (IL-1beta) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1beta release and dissected the signaling pathways leading to LPS-induced IL-1beta secretion. We demonstrate that both the NF-kappaB and the C/EBPbeta-binding elements of the IL-1beta promoter drive LPS-induced IL-1beta gene expression. NF-kappaB activation requires the classical TLR4-initiated signaling cascade leading to IkappaB phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBPbeta activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBPbeta, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBPbeta. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1beta. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1beta gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1beta gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer.     

Stimulation of interleukin-6 release by interleukin-1beta from isolated human adipocytes

The secretion of interleukin-6 (IL-6) is modulated by immune, hormonal and metabolic stimuli in a cell-specific manner. We investigated the effect of cytokines, TNFalpha and IL-1beta, and insulin on IL-6 release from human adipocytes and peripheral blood cells (PBC). Adipocytes released IL-6 constitutively (after 5 h: 5.64 [1.61-15.30]pg ml(-1), after 10 h: 15.95 [2.34-45.59]pg ml(-1), p = 0.007), while PBC secretion did not change significantly over this period. LPS stimulated IL-6 secretion in PBC after 5 h but was without effect on adipocytes. TNFalpha and insulin induced IL-6 production from PBC, but had no effect on adipocytes. IL-1beta, however, induced a substantial increase in IL-6 release in adipocytes and PBC (all p < 0.05). Adipose tissue production of IL-1beta was assessed in vivo by measuring arterio-venous differences across the subcutaneous abdominal adipose bed. Net release of IL-1beta was not observed, suggesting that under basal conditions there is no detectable release of this cytokine into the circulation from this depot. In conclusion (1) PBC demonstrate regulated IL-6 release, while the adipocyte release has a large constitutive component; (2) immune modulators, such as LPS, TNFalpha and IL-1beta, all induce PBC IL-6 release, but only IL-1beta stimulates adipocyte release. Though IL-1beta is not an endocrine signal from adipose tissue, it is an autocrine/paracrine stimulator of IL-6 release from human adipocytes.     

Glyco-tuftsin derivatives modulate interleukin-1 and tumor necrosis factor production

Six Thr1 (O-glyco)-derivatives of the "phagocytosis stimulating peptide" tuftsin, H-Thr-Lys-Pro-Arg-OH and the N-glycosylated undecapeptide H-Thr-Lys-Pro-Arg-Glu-Gln-Gln-Tyr-Asn(beta-D-GlcNAc)-Ser-Thr-OH, which correspond to the "tuftsin-region" at the Fc-domain of immunoglobulin G (amino acid residues 289-299), were evaluated in comparison with tuftsin and rigin, H-Gly-Gln-Pro-Arg-OH, for their capacity to evoke the release of interleukin-1 and tumor necrosis factor from mouse peritoneal macrophages and from human monocytes. Several glycosylated tuftsin derivatives were found to modulate, in a rather dose-dependent manner, the release of the two cytokines from both cell types.     

Adenosine inhibits the release of interleukin-1beta in activated human peripheral mononuclear cells

The effects of adenosine and subtype-specific activators of adenosine receptors (A1, A2A, A2B and A3) were studied on the release of interleukin-1beta (IL-1beta) from peripheral mononuclear cells, monocytes and lymphocytes. In the cells activated by the protein kinase C specific phorbol ester (phorbol 12-myristate 13-acetate) and Ca(2+) ionophore (A23187) both adenosine and the subtype-specific receptor agonists, CPA (A1), CGS 21680 (A2A) and IB-MECA (A3) induced a concentration-dependent inhibition of IL-1beta release. The rank order of potency in the inhibition of IL-1beta release was CPA=CGS 21680>IB-MECA>adenosine>NECA (in the presence of A1, A2A and A3 receptor inhibitors). The inhibitory actions of CPA, CGS 21680 or IB-MECA were significantly reduced in the presence of DPCPX, ZM 243185 or MRS 1191 as subtype-specific antagonists on A1, A2A and A3 adenosine receptors, respectively. It can be concluded that adenosine inhibits the release of IL-1beta from the activated human peripheral mononuclear cells. In this process A1, A2A and A3 receptors are involved.     

Stimulation of ACTH release by human interleukin-1 beta, but not by interleukin-1 alpha, in conscious, freely-moving rats

Ever since two distinct molecules of human interleukin-1 (termed interleukin-1 alpha and interleukin-1 beta) were cloned, sequenced and expressed, it has been a matter of investigation whether these two forms of interleukin-1 possess an identical spectrum of biological activities. Our current studies of interleukin-1 and its involvement in the hypothalamic-pituitary-adrenal axis have indicated that there is a clear-cut differential response to interleukin-1 alpha and interleukin-1 beta. The intravenous injection of human recombinant interleukin-1 beta significantly increased the plasma levels of adrenocorticotropic hormone in a dose-related manner, whereas interleukin-1 alpha did not. This observation suggests for the first time that the two members of the interleukin-1 family may have a different spectrum of biological actions.     

Differential biological activities of human interleukin-1 alpha and interleukin-1 beta

We examined the biological effects induced by both human recombinant interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in five different cell types of human, rat and mouse origin. IL-1 alpha and beta preparations were standardized in terms of biological activity in the EL-4/CTLL bioassay and, in parallel, employed to stimulate PGE2 secretion in human fibroblasts, mesangial cells (MC), C57B1/6 mouse MC, DBA/2 mouse macrophages and Sprague Dawley rat MC. In addition, the co-mitogenic effects of IL-1 alpha and beta were determined in freshly prepared Sprague Dawley rat thymocytes. No significant differences in IL-1 alpha and beta concentration dependent PGE2 production were detectable in the different cell types (MC, fibroblasts and macrophages) of human or mouse origin. Incubation of Sprague Dawley rat MC with both IL-1 alpha and beta resulted in a concentration dependent production of PGE2. However, in contrast to mouse or human MC the potency of IL-1 beta to induce PGE2 in Sprague Dawley rat MC was 26-fold higher compared to IL-1 alpha. In addition, the potency of IL-1 beta to enhance co-stimulated proliferation of Sprague Dawley thymocytes was 200-fold higher than that of equal biological activities of IL-1 alpha. When we tested the additive effects on Sprague Dawley cells, increasing IL-1 beta concentrations added to a fixed IL-1 alpha concentration resulted in a cumulative rise in both, PGE2 secretion by MC and thymocyte proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A point mutation uncouples human interleukin-1 beta biological activity and receptor binding

Interleukin-1 proteins elicit a number of biological activities, but the molecular events following formation of a cell surface receptor-ligand complex have not been well defined. Conversion of Arg127 to Gly127 in the mature human interleukin-1 beta protein reduces bioactivity by 100-fold while the receptor binding affinity decreases by only 25%. The results suggest that the mutant IL-1 beta protein is defective in activating signal transduction events and indicate that binding of interleukin-1 beta protein to receptor is necessary but insufficient for biological activity. The finding that the features of the IL-1 beta protein responsible for receptor binding and biological activity are at least in part distinct may be clinically relevant to the design of interleukin-1 antagonists.     

Crystallization of purified recombinant human interleukin-1 beta

The gene for human interleukin-1 beta was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1 beta) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1 beta +). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on two-dimensional (2-D) gels as a single spot with a pI of 6.7 +/- 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL-1 beta have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P4(1) or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 A resolution. A structure determination based on these crystals is under way.     

Novel indolizine derivatives with unprecedented inhibitory activity on human farnesyltransferase

The rational structural modification of new substituted indolizin-3-yl(phenyl)methanones 1a–i, 2a–i and 3a–i has greatly improved human farnesyltransferase inhibition. The para-bromophenyl analog 2f bearing an ester unit on the indolizine ring demonstrates the highest inhibition potential, with IC₅₀ value of 1.3 ± 0.2 μM. The amidic series 1a–i proves to be the most promising for future modulations, particularly at the triple bond level.     

Interleukin-1 alpha and beta induce interleukin-1 beta gene expression in human dermal fibroblasts

The ability of the two forms of interleukin-1, IL-1 alpha and IL-1 beta, to induce IL-1 beta gene expression in human skin fibroblasts was studied in vitro, using Northern blot hybridization. Both recombinant IL-1 alpha and IL-1 beta caused a dramatic increase in IL-1 beta mRNA levels, IL-1 alpha being more efficient than IL-1 beta. Blockage of the prostaglandin synthesis by indomethacin reduced the basal level of IL-1 beta mRNA in control cultures and decreased also the stimulatory effect exerted by both IL-1s on IL-1 beta gene expression. These data suggest that IL-1 and prostaglandin (mainly PGE2) may act synergistically to stimulate IL-1 gene expression in dermal fibroblasts, contributing as a local amplifier system to the alterations of connective tissue in inflammatory processes.     

Associations of interleukin-6 with interleukin-1beta, interleukin-8 and macrophage inflammatory protein-1beta in midlife women

Objective: The aim of the present study was to determine the associations of interleukin (IL)-6 with other cytokines and chemokines and to compare these associations in peri- and postmenopausal women. Methods: Ninety-nine perimenopausal and 92 postmenopausal women were enrolled in this study. Serum concentrations of IL-6, IL-1β, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, tumor necrosis factor (TNF)-α, interferon γ, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, macrophage inflammatory protein (MIP)-1β and monocyte chemotactic protein (MCP)-1 were measured simultaneously using a multiplexed cytokine assay. Results: Among the 17 cytokines, IL-6, IL-1β, IL-5, IL-7, IL-8, IL-10, MCP-1 and MIP-1β were detected in serum in more than 50% of the women. Serum levels of IL-4 and MCP-1 in postmenopausal women were significantly higher than those in perimenopausal women. Serum IL-6 concentrations showed significant and positive correlations with serum concentrations of IL-1β, IL-8, MIP-1β, IL-7 and MCP-1 in women regardless of menopausal status, and these correlations were still significant after adjustment for age and body mass index. Conclusion: Serum IL-6 concentration was found to be closely associated with serum concentrations of IL-1β, IL-8, MIP-1β, IL-7 and MCP-1 in women regardless of menopausal status, suggesting that these cytokines act in concert with the progression of several symptoms and various diseases.     

Extracellular human thioredoxin-1 inhibits lipopolysaccharide-induced interleukin-1beta expression in human monocyte-derived macrophages

Oxidative stress plays an important role in atherosclerotic vascular disease, and several recent studies were focused on thioredoxin-1 (Trx-1) and its potential protective role against oxidative stress. Since human monocyte-derived macrophages (HMDM) are important cells in several inflammatory diseases including atherosclerosis, we conducted this study to evaluate the impact of extracellular recombinant human Trx-1 (rhTrx-1) on gene expression in lipopolysaccharide-activated HMDM. Our results showed that rhTrx-1 was capable of reducing interleukin (IL)-1beta mRNA and protein synthesis in a dose-dependent manner. This effect was partly mediated through a reduction of NF-kappaB activation as analyzed by transient transfection and gel shift assays. In addition, we showed that the attenuation of NF-kappaB activity was the result of the reduction of both p50 and p65 subunit mRNA and protein synthesis on one hand and of the induction of I-kappaBalpha mRNA and protein expression on the other hand. Moreover, inhibition of endogenous Trx-1 mRNA was also observed, suggesting a contribution to the diminution of NF-kappaB activity since endogenous Trx-1, in contrast to the exogenous Trx-1, activates the NF-kappaB system. Finally, H2O2-oxidized rhTrx-1 reduced IL-1beta mRNA synthesis in lipopolysaccharide-activated HMDM. This result highly suggested that the rhTrx-1 used in this study could be oxidized in the culture medium and, in turn, reduced IL-1beta mRNA and protein synthesis. Taken together, these data indicated a potential new mechanism through which extracellular rhTrx-1 exerts an anti-inflammatory function in HMDM.     

Multiple charge isomers of human recombinant interleukin-1 beta

Human interleukin-1 beta (rhuIL-1 beta), obtained by DNA recombinant technology, was radiolabelled. Its isoelectric properties were determined by various analytical techniques such as high-voltage ultrathin layer isoelectric focusing (IEF) and chromatofocusing. The rhuIL-1 beta molecule had a molecular mass of 18 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. When examined by IEF on a polyacrylamide gel of 1 mm thickness in the pH range of 3.5-9.5, it was resolved into two broad bands appearing in the pH range of 6.2-5.8 and 5.5-5.2. Each of the two bands was further resolved into multiple bands when electrofocused on (i) a thinner gel of 0.5 mm thickness and (ii) a narrower pH range of 5-8. Upon chromatofocusing in a liquid column, it was possible to isolate various charged components of rhuIL-1 beta. However, all these components reacted to the antiserum to rhuIL-1 beta and displayed a molecular mass of 18 kDa suggesting the charge heterogeneity of rhuIL-1 beta.     

Purification and characterization of human recombinant interleukin-1 beta

A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.     

Radioprotection by N-palmitoylated nonapeptide of human interleukin-1beta

Interleukin-1beta (IL-1beta) is a cytokine involved in homeostatic processes of the immune system and specifically in inflammatory reactions. The nonapeptide of human IL-1beta (VQGEESNDK, position 163-171) has been shown to retain adjuvant and immunostimulatory activities of the native molecule without any inflammatory and pyrogenic properties. A lipophilic derivative of IL-1beta nonapeptide having a palmitoyl residue at the amino terminus was synthesized in order to determine the effects of such structural modification on its bioactivities. The structurally modified peptide derivative, palmitoylated peptide, significantly protected C3H/HeN mice against potentially lethal doses of ionizing radiation. The dose reduction factor was found to be 1.07. Hematological studies show improved recovery of red blood cells and platelets in irradiated and palmitoylated peptide treated mice as compared with the untreated and irradiated group. These results suggest the importance of the derivatization of small peptides of radioprotective, but toxic cytokines in order to enhance radioprotective activity while reducing unwanted toxic side effects.     

Signal transduction via the beta 1 integrins is a required intermediate in interleukin-1 beta induction of alkaline phosphatase activity in human osteosarcoma cells

In this report data are presented which demonstrate that the induction of an osteoblastic phenotype by interleukin-1 beta (IL-1 beta) requires an intermediate step involving signal transduction via the beta 1 family of integrin glycoproteins. Recombinant human IL-1 beta inhibits human osteosarcoma cell proliferation, stimulates integrin expression, and induces alkaline phosphatase activity, a marker of osteoinductive and osteoblastic phenotype. The approximately 10-fold stimulation of expression of the beta 1 integrins occurs rapidly (within 20 to 40 h), whereas the alkaline phosphatase activity is not induced until at least 5 days after the addition of IL-1 beta. To determine whether the early stimulation of integrin expression is required for the subsequent expression of alkaline phosphatase activity, polyclonal as well as monoclonal antibodies directed against the alpha 5 and beta 1 integrin subunits were added to cultures at the same time as IL-1 beta. These antibodies inhibited by 55 to 82% the longer term induction of the osteoblastic differentiation marker, alkaline phosphatase activity, but did not however affect the IL-1 beta-induced stimulation of integrin expression or the inhibition of cell proliferation. In addition, at the concentrations used, there was no effect of the antibodies on cell attachment. These data suggest that the stimulation of integrin expression by IL-1 beta, and the resulting enhanced integrin-extracellular matrix interactions, is a required intermediate event in the IL-1 beta regulation of osteoblastic cell differentiation. The data also suggest that the integrins are capable of signal transduction resulting in altered gene expression, and may also play a crucial role in modulating cytokine-mediated effects on cell differentiation.     

A short synthetic peptide fragment of human interleukin-1 beta increases both human and murine natural killer activity

The effect of a short synthetic fragment of human interleukin-1 beta (hu IL-1 beta) on natural killer (NK) activity was examined. Peripheral-blood mononuclear cells (PBMC) from normal donors showed a significant increase in NK activity against K562 leukemia cells after preincubation for 18 h with the IL-1 peptide. A similar augmentation was not observed after culturing the cells in the presence of hu IL-1 beta. The increase in tumor cell lysis could not be ascribed to a cytolytic activity of the synthetic fragment on target cells, since the peptide caused no direct lysis of various tumor cell lines. Although the peptide enhanced NK cytotoxicity of PBMC, highly purified large granular lymphocytes were not susceptible to its stimulatory effect. The addition to the cultures of antibodies to human interleukin-2 (hu IL-2) completely blocked the peptide-induced boost of NK cytotoxicity, suggesting that IL-2 is mainly involved in the activation process. The ability of the IL-1 peptide to increase NK activity was further confirmed in vivo in the mouse. Cytotoxicity against YAC-1 lymphoma cells, which was very low in the spleen of untreated BALB/c mice, was in fact significantly increased after a single inoculation of the peptide. These data thus indicate that a short synthetic peptide fragment of hu IL-1 beta is able to increase both human and murine NK activity.